Acetylcholine
(ACh) receptors in the mammalian CNS can be divided into muscarinic (mAChR)
and nicotinic (nAChR) subtypes based on the ability of the natural alkaloids,
muscarine and nicotine, to mimic the effects of ACh as a neurotransmitter.
Until recently, studies of the neuropsychopharmacological effects of ACh have
focused on mAChRs, while nAChRs have been evaluated primarily for their role
in mediating neuromuscular and autonomic transmission.However, over the last decade, this trend has changed following
preclinical and clinical studies indicating that neuronal nAChRs may have
a substantial role in mediating antinociception, cognitive performance, modulating
affect, and enhancing the release of other neurotransmitters.

The
majority of evidence defining potential therapeutic targets involving neuronal
nAChRs has resulted from studies on the effects of (-)-nicotine in a variety
of preclinical and, to a lesser extent, clinical models.And, while a significant number of neuronal nAChR receptor subtypes
have been potentially identified based on subunit structure at the molecular
level, little is known regarding the physiological role of most of these receptor
subtypes beyond what can be deduced from their discrete localization within
brain tissue. The development of receptor subtype selective ligands, especially
antagonists, may be anticipated to facilitate the definition of receptor subtype
function.In this chapter the molecular
biology of neuronal nAChRs is discussed within the context of the pharmacology
of agonists, antagonists and allosteric modulators.In addition, the potential CNS therapeutic targets for nAChRs are reviewed.

Classification
of nAChRs

Workers
at the Salk Institute and at the Institut Pasteur have established that the
muscle nAChR is a ligand-gated ion channel (LGIC) receptor composed of 5 subunits—two
a1 subunits, and one each of b1, d and g (or e, depending on the stage of development)
[14].Several genes
have been identified in rat and chick neural or sensory tissue that encode
for neuronal nAChR subunits that are distinct from those in the muscle nAChR,
providing for a multitude of potential subtypes of neuronal nAChRs.The wide distribution of the some of these
transcripts in mammalian brain indicates that neuronal nAChRs represent a
major neurotransmitter receptor superfamily related to other LGICs including
serotonin (5HT3), GABAA, N-methyl-D-aspartate (NMDA),
and glycine.However, in contrast
to these other LGICs where established pharmacology rapidly segued into the
molecular biology, the pharmacology of neuronal nAChRs has only started to
emerge as a result of the rapid advances in the molecular biology of the nAChR
family.

Receptor
nomenclature in the nAChR area has been derived from classical pharmacology
approaches, including receptor sensitivity to snake toxins.Following from Dale's conceptualization of
ACh receptor subtypes over 80 years ago, Barlow and Ing and Paton and Zaimis
showed that the antagonist decamethonium (C10) was more effective than hexamethonium
(C6) in blocking muscle nAChRs, whereas C6 was effective in autonomic ganglia
[83].This led to
the description of 'C10 ' (muscle) and 'C6' (neuronal) receptors.Muscle nAChRs are selectively activated by
phenyltrimethylammonium and are pseudo-irreversibly blocked by a-bungarotoxin (a-BgT).Ganglionic nAChRs are preferentially activated
by 1,1-dimethyl-4-phenylpiperazinium (DMPP), competitively blocked by trimethaphan,
and are resistant to snake a-toxins, yet sensitive to neuronal bungarotoxin
(n-BgT: also known as k-BgT, a-BgT 3.1, or toxin F).

In
mammalian brain, two major neuronal nAChR subclasses can be delineated using
radioligand binding [17]: those that bind a-BgT with high affinity (a-BgT-sensitive nAChRs (Kd ~ 0.5 nM using [125I]a-BgT) and those that do not
(a-BgT-insensitive nAChRs).a-BgT-sensitive nAChRs have
low affinity for (-)-nicotine, whereas a-BgT-insensitive nAChRs have
high affinity (Kd = 0.5 - 5 nM) for [3H](-)-nicotine, [3H]ACh,
[3H]methylcarbamylcholine (MCC), and [3H]cytisine).All four of these [3H]agonist ligands
are thought to interact with the same ACh binding sites on the nAChR.

Molecular
Diversity of nAChRs

nAChRs
are encoded by a family of related but distinct genes that share a common
origin and have a long phylogenetic history.To date eleven neuronal nAChR subunits have been described;Eight (a2 - a9) code for a subunits (see [7,14] for reviews) based on the presence of adjacent cysteine
residues in the predicted protein sequences, in a region homologous to the
putative agonist binding site of the muscle a subunit (a1) while three are referred
to asnon-a or b-subunits(b2-b4).Each of the nAChR subunits
displays a characteristic phenotype of structural features extending from
the N-terminus to the C-terminus (Figure 1):
(1) a large (~200 amino acids) N-terminal hydrophilic domaincontaining the multiple loops of the neurotransmitter binding site;
(2) the highly variable C-terminal hydrophilic domain that faces the cytoplasm,
where it can be phosphorylated; and (3) a set of four closely spaced transmembrane
domains—termed M1-M4—immediately following the large extracellular domain.The M2 domain is believed to form the wall
of the ion channel.The amino acid
sequences of the neuronal nAChR genes except a7 and a8 are between 40 - 60% similar
[7,49].The a7 and a8 genes share approximately
70% similarity, with a much lower level (<30%) of similarity to the other
nAChR genes.The a9 subunit is the most unique
of the a-subunits displaying less than
50% similarity to a2-a8[26].Comparison
of the nAChR sequences also reveals that several pairs of subunits display
homologies that are instructive in considering their function.For example the b2 and b4 are highly homologous, consistent
with their ability to substitute for one another in forming functional channels
when paired with a2, a3, or a4 subunits (see below).The similarities in sequence between a6 and a3 may have caused misidentification
of a6 subunits as a3 subunits in early localization studies on
the a3 subunit ([41], but see [87]).

The
chromosomal localizations of many of the human neuronal nAChR genes have been
determined [34]. The human a4 gene is located on chromosome
20 and is associated with an autosomal dominant nocturnal frontal lobe epilepsy
[44,49]. The human a7 gene has been mapped to chromosome 15 and has
been associated with some forms of schizophrenia [46].Additionally,
the human a3, a5 and b4 are tightly linked on chromosome
15 while the a2 and b3 genes are found on chromosome 8 [34].

Although
all neuronal nAChRs are pentamers, the structure and stoichiometry of many
neuronal nAChRs remains to be fully defined.The a7, a8 and a9 subunits can form functional pentameric homo-oligomers while the stoichiometry
of a4b2 nAChRs consists of two a4 and three b2 subunits [7].It is believed
that pairwise expression of a2, a3, and perhaps a6 subunits with either b2 or b4 subunits results in similar
stoichiometries.More complex combinations
containing three (a3b4a5) or four subunits (a3b2b4a5) have been immunoisolated from brain [49].Site-directed
mutagenesis of affinity labelled residues in the channel and binding site
domains have demonstrated that mutations of single amino acids can modify
multiple functions of the nAChR [14].For instance,
mutations in the M2 channel lining domain of the a7 subunit can produce either
a significant increase in apparent affinity for agonists or a loss of desensitization
and a conversion of competitive antagonists to agonists [14].

CNS Expression
of Neuronal nAChRs

Both
a-BgT-sensitive and a-BgT-insensitive nAChRs have been extensively
mapped in rodent brain and, to some extent, in human brain.Using radioligand binding and in situ hybridization, the topographical
distribution of nAChRs corresponds well with the effects elicited by (-)-nicotine
and the known functions associated with each brain region.

In
rat, high affinity nAChR sites revealed by [3H](-)-nicotine, are
abundant in selective areas of the cerebral cortex (predominantly layers III
& IV ), thalamus, interpeduncular nucleus and the superior colliculus,
but are of low to moderate abundance in the hippocampus and hypothalamus (Table
1).The second class of sites,
labeled by [125I] a-BgT, are enriched in the hippocampus, hypothalamus and layers I and VI
of the cerebral cortex [17].

In situ hybridization assays have demonstrated
that the a4 subunit mRNA is expressed strongly in a number of areas including the
thalamus, deeper layers of the cerebral cortex, ventral tegmental area (VTA),
the medial habenula and substantia nigra (SN) pars compacta [44] and that a3 is expressed strongly in
the locus coeruleus (LC), habenula, and interpeduncular nucleus [87].These subunits
are also expressed in peripheral tissue, with a3 being expressed in autonomic and sensory ganglia
and a4 being expressed in the trigeminal
ganglion [28].The a2 subunit has a much more restricted pattern
of expression, with mRNA being expressed at high levels only in parts of the
interpeduncular nucleus [44].a7 message is particularly high in the hippocampal
formation, which correlates well with the high level of a-BgT binding this region [44].a8 has not been found in rat and the distribution
of a9 appears restricted to skin
and sensory tissue.The presence of
a9 subunits in nAChRs in cholinergically-innervated
outer hair cells of the cochlea may explain the unusual cholinergic pharmacology
found in this tissue [59].Among the b subunits, b2 is widely expressed, whereas
expression of b4 and b3 is more variable [44].b4 message is highly expressed in a number of
areas in which substantial a3 message is found, including
the habenula, area postrema, and LC [87].One notable
exception to this pattern is that substantial expression of a3 message is found in DA neurons in the SN,
whereas b4 message is absent [87].b3 message appears to be prominent in brainstem
catecholaminergic areas such as SN and LC [44].Similarities
in the distributions of a6 and b3 message suggest that these two subunits might
be important in the nAChR-mediated effects on catecholamine release [44], although the lack of pharmacological tools selective
for nAChR subtypes containing these subunits has restricted investigation
of the pharmacology of this effect (see below).

Localization
of mRNA or binding sites to particular brain nuclei does not reveal which
cell types in the identified regions express nAChR subunits.Interest in this more fine-grained analysis
has accelerated of late, although information is still somewhat sparse.Dopamine neurons in the SN appear to express
the a3 subunit but not the b4 subunit, since neurons labeled by an antibody
for tyrosine hydoxylase in the SN express mRNA for a3, but not b4.Moreover, tyrosine hydroxylase-positive neurons in the SN and the
VTA can also immunostained by an antibody for the a6 subunit [32].Similarly,
double labeling experiments suggest that serotonergic neurons in the brainstem
express the a4 subunit [6].

Much
less is known about the expression of nAChR subunit genes in human brain (for
review see [34]).In humans
high densities of high affinity nicotine binding sites are found in some areas
that are also rich in a4 message in rats, such as
SN (pars compacta) and thalamus; and a7 message and a-BgT binding are prominent in the hippocampal
formation of both rats and humans [8,34].Differences
do exist in the regional distribution of these subunits across species, but
differences in the post-mortem handling of tissue from these species may account
for some of these discrepancies [8].Whether interspeciesdifferences in subunit distribution translate into differences in pharmacological
properties across species will require further investigation.

The
co-existence of different nAChR subunits in the same central or peripheral
nervous system pathway (and even in the same neuron) has made the in vivo study of the properties of individualnAChR subtypes extremely difficult.For this reason, heterologous expression studies in either Xenopus
oocytes or mammalian cell lines haveprovided considerable information on the electrophysiological and pharmacological
properties of different subunit combinations.

Functional
responses occur in oocytes or cell lines transfected with pairwise combinations
of rodent, avian or human a and b subunits, confirming biochemical findings which suggest that many native
nAChRs consist of a/b heteromers [15,34].These heterologous expression studies
have demonstrated that both the a and b subunits determine the functional and pharmacological
properties ofthe subunit combination.For example, when expressed with the b2 subunit, the a2, a3 and a4 subunits all form functional channels that
differ in their channel open times, single channel conductance and agonist/antagonist
sensitivity. However, not all subunit combinations form functional nAChRs.The b3 and a5 subunits are unable to form
functional nAChRs when expressed alone or in pairs with other subunits but
instead appear to act as “regulatory” subunits [34].For example,
in combination with the human a3 and b2 or b4 subunits, a5 increases the desensitization rate, and in
the case of the a3b2 combination, a5 significantly alters the
EC50 values for nicotine and ACh.It may be that the a5 subunit occupies a position
equivalent to the b1 subunit of muscle nAChRs
so that it interfaces with the surfaces of adjacent subunits that are incapable
of forming ACh-binding sites [86].

Studies
of the single channel properties of neuronal nAChRs expressed in oocytes indicate
considerable diversity among heterologously expressed subunit combinations
[62]. For example, two distinct populations of open channel
conductances can be observed after injection of either a2b2 or a3b2 subunits into oocytes. In
contrast, the a4b2 subunit combination generates only a single type of channel.Of the b2 - containing receptors, the current of the a3b2 receptors is the most sustained
while the a2b2 combination gives the greatest peak current. nAChRs
containing the b2 subunit
are thus likely to generate brief synaptic currents in vivo, creating the potential for rapid
signal processing.In contrast, currents
for the a3b4 subunit combination are of a smaller conductance but do not desensitize
as rapidly.Accordingly, if a3b4 receptors
predominate at synapses, responses may be prolonged, providing more time to
organize/integrate a cellular response.

a7,a8and a9 gene products differ from other members of
the nAChR superfamily in that they can form functional receptors in oocytes
when expressed as homo-oligomers [7,34]. The most striking pharmacological characteristic of
the a7 homo-oligomeric
receptor is its marked permeability to calcium ions [1].This finding,
coupled with the unique distribution pattern of this receptor in brain [17] has led to heightened interest in the a7 subtype as a potential therapeutic
target.It is noteworthy that most
of the structures with the highest abundance of a7 transcripts in rodents are
major components of the limbic system.A
role for this receptor subtype in the regulation of neurite outgrowth and
survival has also been suggested [25].

Considerable
progress has been made in the understanding of the pharmacological diversity
of nAChRs based on the availability ofherterologous
expression systems expressing various a/b subunit combinations.The rank order of potency of four nAChR activators,
ACh, (-)-nicotine,(-)-cytisine,
and DMPP on receptors formed from human b2 or b4 subunits in combination with
a2, a3, or
a4 subunits
in Xenopus oocytes, has established the importance of both a - and b-subunits in defining the pharmacological
properties of the nAChR with these distinct subunit combinations ([15]; Table 2).Cytisine was the least efficacious agonist at AChRs containing b2 subunits but it displayed significant activity
at b4 containing subunits.ACh was the most efficacious agonist at all
AChRs except a3b2 but was among the least potent of the agonists
at all nAChR subunit combinations.While
studies using cell lines stably expressing these same human nAChR subunit
combinations have revealed a similar rank order of potency for these agonists
[33,78], some important differences in either potency and/or
efficacy were observed.The pharmacological
differences detected in Xenopus oocytes and cell lines stably expressing defined
human nAChR subunit combinations may result from differences in the methods
used to analyze function (electrophysiology versus ion flux), differences
in subunit stoichiometries, or differences between the two expression systems.Additionally, significant pharmacological differences in the agonist
potencies and sensitivities between the oocyte-expressed a3b2 and a3b4 human subtypes and the same
rat and/or chick subtype have been observed [34].For example,
(-)-nicotine is more potent than ACh at the human a3b2 subtype but it is less potent
than ACh at the rat subtype.These
species differences suggest that ascribing functional/behavioral effects of
nAChR ligands to a specific rodent subtype(s) based on data generated using
human nAChRs expressed in heterologous expression systems may not be valid.

The
homomeric a7- a9 subunits expressed as homo- oligomers in oocytes also form functional
cation channels gated by nicotinic agonists with differing pharmacological
properties. The agonist sensitivities of chick a7 and a8 expressed in oocytes show
that the a8 homomers exhibit higher affinity for nicotinic agonists as compared to
a7 homomers.The order of potency for a8 was (-)-nicotine = (-)-cytisine
(EC50 = 1 µM) ~ ACh > DMPP > tetramethylammonium.Incontrast, DMPP is a very weak partial agonist
for a7 and tetramethylammonium has no effect. a9 nAChRs expressed in vitro exhibit the most unique pharmacological
properties identified to date.(-)-Nicotine,
atropine, strychine, d-tubocurare and muscarine all behave as antagonists
while DMPP acts as an agonist [26].

While
studies in oocytes and cell lines have yielded some clues as to the physiological/pharmacological
roles of the different nAChR subunits in vivo, some caution is required in interpreting
these findings because of the atypical nature of the host cell environment.In addition, the expression of multiple nicotinic genes in central
and peripheral tissues suggests that some nAChR subtypes in these areas may
contain more than two types of subunit [84], a finding that would explain why some of the pharmacological
properties of receptors formed by injection of a single subunit (a7) or
the pairwise combination of a/b subunits do not correlate well with the properties of receptors found
in neurons [18,50].Despite the
lack of precise correspondence between nAChRs in these in vitro expression systems and native receptors, pharmacological
data derived from oocyte studies have been useful in characterizing native
nAChRs.For example, three distinct
hippocampal nAChRs identified electrophysiologically were designated as a7-containing, b2-containing, and b4-containing based on the similarities between
the characteristics of these native receptors and characteristics of a7-containing, b2-containing, and b4-containing nAChRs expressed in oocytes [1].The accuracy
of this designation has recently been supported by the demonstration that
only the current originally believed to be mediated by a b2-containing receptor is absent in b2-knockout mice, and only the current believed
to be mediated by an a7-containing receptor is missing
in a7-knockout mice [61,89].

Information
from heterologous expression studies combined with in situ hybridization studies has identified intact in vitro model systems in which the pharmacology of compounds acting at putative
subtypes of neuronal nAChRs can be evaluated using electrophysiological and
biochemical techniques.For example, the a4b2 subunit isoform appears to modulate the flux of monovalent ions as measured
by efflux of [86Rb+] from thalamic synaptosomes [54] and is believed to play a role in the release of ACh
from rat hippocampus (see below).

Thus,
heterologous expression studies have started to refine the combinatorial diversity
of neuronal nAChR subunits suggested by the initial cloning and expression
studies.A significant challenge in
the years to come will be to relate the properties of nAChRs recorded in
vivo at the level of a particular neuronal pathway, with a defined homo-oligomeric
or hetero-oligomeric subunit combination.Such an understanding may pave the way to “circuit-targeted” molecular
pharmacology and drug discovery [14].

Functionally
Distinct Transition States of nAChRs

In
addition to structurally distinct subtypes of nAChRs, there are functionally
distinct transition states for an individual nAChR.Current evidence regarding the states of activation and desensitization
of nAChRs derives primarily from work on the muscle and a7 nAChRs [14].Distinct ligand
binding sites, some sensitive to ACh and (-)-nicotine and others involving
distinct classes of allosteric modulator sites on, and between, the various
receptor subunits, can cooperatively modify, either positively or negatively,
the equilibrium between the receptor states affecting the proportion of receptors
existing in each state but not significantly altering the intrinsic binding
and physiological properties of the states themselves.Thus the nAChR functions within the context
of the classical allosteric “concerted scheme” [12,14] for oligomeric proteins that incorporates the multiple
state concept originally proposed by Katz and Thesleff for the nAChR.

The
allosteric transition state model considers a minimum of four interconvertible
states with differing rates of interconversion: a resting state (R); an activated
state (A) with the channel opening in the ms to ms timescale and having
low affinity (mM to mM) for agonists; and two 'desensitized' closed channel states (I
or D) that are refractory to activation on a ms (I) to minute (D) timescale
but exhibit a high affinity (pM - nM) for nAChR agonists and some antagonists.nAChR ligands may therefore be considered to
differentially stabilize the conformational states to which they preferentially
bind.

A
more persistent modulation of nAChR function can occur by phosphorylation
of the receptor protein [85].While little
is known regarding phosphorylation of neuronal nAChRs, the sites of phosphorylation
and the associated protein kinases have been well characterized in the Torpedoreceptor.At least four kinases differentially phosphorylate muscle and Torpedo nAChR subunits: cAMP-dependent
kinase (PKA); protein kinase C (PKC), which also phosphorylates the neuronal
receptor; a tyrosine kinase; and a Ca2+ -calmodulin kinase.Phosphorylation can enhance the rate of nAChR
desensitization and increase the frequency of spontaneous channel openings.

Sites of
nAChR-Ligand Interaction

Evidence
is rapidly emerging to indicate that the nAChR channel may be activated through
sites distinct from the classical ACh binding sites and suggests that “cholinergic
channel modulators” (ChCMs) may be a more appropriate, and all encompassing,
classification for those compounds that activate, inhibit or desensitize nAChRs
[10].

The ACh binding
site. Binding
site(s) for cholinergic ligands on the nAChR were initially thought to reside
solely on the a subunit. More recently, site - directed mutagenesis studies have shown
that binding sites for cholinergic ligands on nAChR are located at the interfaces
between the a and b subunits in heteromeric receptors and between a subunits in homomeric receptors [14].For example,
a4-containing and a3-containing neuronal nAChRs differ dramatically
in their sensitivity to nicotinic agonists and antagonists.Analysis of chimeric subunits consisting of
portions of these two a subunits have indicated that the region from the amino terminus to position
84 is important in determining sensitivity to the agonists, ACh and(-)-nicotine, positions 84 to 121 and from position 121 to 181 contain
amino acid residues important in determining n-BgT - sensitivity, while the
sequence segment from position 195 to 215 is important for both agonist and
antagonist sensitivity.In particular, the amino acid residue at position
198 (glutamine in a3 and proline in a2) is believed to be important in determining the sensitivity of neuronal
nAChRs. In the case of the a4 subunit, amino acids 151-155
and 183-191 have been found to confer the physiological and pharmacological
properties typical of the a4b2 receptor [19].

AlternativeChannel "Activator" sites.Neuronal nAChR function may
also be enhanced via ligand binding sites distinct from those at which ACh
or (-)-nicotine interact. These sites are thought to be present at the level
of the a subunit
and are not subject to the same desensitization mechanisms described for (-)-nicotine.Compounds that interact with this novel site to increase neuronal nAChR
mediated ion conductance have been termed “channel activators.”The cholinesterase inhibitors physostigmine and galanthamine, (+)-2-methylpiperidine,
and the antihelminthic agent, ivermectin, are examples of compounds that act
as channel activators at this site which is distinct from the (-)-nicotine
site [1,12].Ivermectin
(30 mM) has been shown to enhance ACh-evoked current
in chick or human a7 nAChRs.The concomitant increase
in apparent affinity and cooperativity of the ACh dose-response curve suggests
that ivermectin acts as a positive allosteric effector of this subtype [12].

Alternative ligand-binding
sites that modulate nAChR function. Based primarily on work from
the muscle nAChR, and supported by preliminary work from the neuronal nAChR,
there is evidence to indicate that there are a number of other ligand-binding
sites that can modulate neuronal nAChR function.

Non-competitive (negative allosteric
modulators) blockers.A number of chemically diverse
molecules, including mecamylamine (Fig. 2),
histrionicotoxin, chlorpromazine, phencyclidine (PCP), MK 801, local anesthetics,
lipophilic agents such as detergents, fatty acids, barbiturates, volatile
anesthetics, and n-alcohols can modify the properties of the nAChR without
interacting with the ACh binding site, or directly affecting the binding of
ACh.

These
non-competitive blockers (NCBs) interact with at least two distinct sites
that differ from those of the competitive blockers. The first site binds ligands
in the low micromolar range, is found within the pore and composed of amino
acids in the M2 segments of the five subunits. Binding of NCBs is facilitated
by agonist binding.Single channel
experiments suggest that interaction at this site causes either a rapid reversible
channel blockade or simply shortens channel opening times in a voltage-sensitive
manner [43]. A second low affinity site has a distinct pharmacology
in that NCBs accelerate desensitization of the nAChR by shifting the equilibrium
towards the desensitized state [43]. Since the ligands to these sites are generally lipophilic
and the number of sites calculated per receptor in reconstitution experiments
depends on the lipid-to-protein ratio, it has been suggested that these sites
lie at the interface between the nAChR protein and membrane lipids.

Steroidbinding sites. Steroids can modulate neuronal
nAChRs expressed in oocytes, chromaffin cells and in brain.This is not surprising considering the clinical
effect of the steroid-like, neuromuscular blocking agent, pancuronium.Steroids are thought to act at an allosteric site distinct from both
the ACh binding site and the ion channel.Progesterone and testosterone, but not cholesterol or pregnenolone,
inhibit in a voltage-insensitive manner, the a4b2,
a3b2 and a7 nAChRs [12]. In chromaffin cells, dexamethasone, hydrocortisone
and prednisolone behave as non-competitive inhibitors of the nAChRs, and in vivothere is an intriguing association between circulating corticosteroids,
[125I]a-BgT binding proteins, and behavioral sensitivity to (-)-nicotine [63].Adrenalectomy
results in corticosterone-reversible increases in the sensitivity to(-)-nicotine in a variety of behavioral and physiological tests in
mice, and chronic corticosterone selectively reduces the density of[125I]a-BgTnAChRs. In vitrocorticosterone (high mM concentrations) inhibited binding of [125I]a-BgT to rat brain membranes
and reduced the affinity of (-)-nicotine for this binding site, which is consistent
with a negative allosteric interaction. Estradiol has been found to differentially
modulate different nAChRs subtypes.While this agent potentiates the responses to ACh at a4b2 nAChRs expressed in oocytes,
it inhibits the effects of ACh at the a3b2 subtype [12].The subtype-specific
ability to activate or inhibit nAChR function with a single allosteric modulator
suggests the potential of targeting the steroid site for drug discovery.

Calcium modulation. The neuronal nAChR has substantial influence on Ca2+ dynamics
neurons by virtue of the permeability of their associated channels to Ca2+.Interestingly, nAChRs may also be a target at clinically relevant (low
micromolar) concentrations of dihydropyridine Ca-blockers like nimodipine
[51] and are also influenced directly by extracellular Ca2+.This latter effect appears to be mediated by
a binding site on the nAChR complex that is distinct from the agonist binding
site [1].Thus, compounds
that affect the dynamics of Ca2+ flux may also indirectly affect
nAChR function.

Antagonists.Neurotoxins are commonly used
to distinguish between neuronal nAChR receptor subunit combinations [52].Lophotoxins
are a family of related neurotoxins isolated from marine soft coral that non-discriminantly
inhibit both neuronal and muscle subtypes of nAChRs.Neosurugatoxin (NSTX), isolated from the Japanese
ivory mollusc (Babyloni japonica)
exerts potent blocking action in autonomic ganglia, antagonizes (-)-nicotine-induced
antinociception in mice, inhibits (-)-nicotine-evoked release of [3H]
dopamine from rat striatal synaptosomes, and blocks ACh- elicited currents
in oocytes containing a2b2, a4b2, and a3b2, but not a7 and a1b1dgnAChR subtypes.The rat
and chick a7 gene expressed as a homo-oligomer in oocytes is highly sensitive to a-BgT and ACh-gated currents
can be completely blocked by nanomolar concentrations of this toxin.Neuronal bungarotoxin (n-BgT) completely blocks
ACh-induced currents in oocytes injected with a3b2 and partially blocks the a4b2 subtype but does not modulate
a2b2 and a3b4 function.The alkaloids dihydro-b-erythroidine (Fig.
2) and erysodine are competitive nAChR antagonists that appear to display
some selectivity for b2 containing nAChRs particularly
the a4b2 subtype [21].Purified from
the Conus magus snail venom, a-Conotoxin-MII inhibits the a3b2 subtype with an IC50
of 0.5 nM, whereas it is from two to four orders of magnitude less potent
at other nAChR subtypes [13].Methyllycaconitine
(MLA, Fig. 2), isolated from the plant,
Delphinium brownii, is a very potent (Ki=
1 nM) inhibitor of [125I] a-BgT binding in rat forebrain preparations,
produces a potent reversible blockade of a7, is >30-fold less potent at the a3b2 or a4b2, and is inactive at the muscle
nAChR [88].Thus, MLA clearly
differentiates between BgT sensitive sites on neuronal and muscle nAChRs.It is anticipated that highly selective antagonists for the different
nAChR subtypes that can readily penetrate the blood brain barrier will become
available in the next few years.Such
agents will greatly aid the ability to correlate in
vitro functional selectivity and in
vivo activity.

Agonists.The last few years have seen
a flurry of medicinal chemistry activity targeted towards the identification
of compounds that activate different nAChR subtypes for the potential treatment
of avariety of disorders (see below).While agonists have been identified that display subtype selectivity
in radioligand binding studies, there are currently no agonist ligands that
potently and selectively activate themajor
nAChR subtypes in in vitro functional assays.A more complete overview of the “classical”
agonists (e.g. nicotine and cytisine) can be found in several recent reviews
[10,22,34,35].The following
discussion is focused on the newer agonists that have appeared in the literature
in recent years (see Fig. 3).

Epibatidine,
a chloropyridine natural product isolated from the venom of the “poison arrow”
frog (Epipedobates tricolor), is
among the most potent nAChR ligands identified to date [3], with a Ki value of 40 pM at the a4b2 nAChR and 20 nM at the a7 subtype.Both of the isomers of epibatidine are potent
full agonists of a4b2, a3b2, a3b4, a7 and a8 nAChRs.Epibatidine also exhibits high potency and
efficacy in activating muscle and ganglionic (a3b4a5?) type nAChRs.The observation that epibatidine has potent
analgesic properties prompted considerable interest in identifying cholinergic
channel modulators lacking the side-effect liabilities of epibatidine as analgesic
agents [3].

ABT-594,
a 3-pyridyl ether, is a centrally acting nAChR agonist with potent antinociceptive
and anxiolytic-like effects in rodent models [23].In vitro functional assays for the human
a4b2,
a7 and abdg nAChR subtypes indicate that
ABT-594 is a full agonist at these subtypes but displays enhanced selectivity
for the a4b2 subtype relative to the a7 and abdg subtypes relative to epibatidine.This enhanced in vitro selectivity supports in vivo studies demonstrating an improved
separation between analgesic effects and side-effects for ABT-594 compared
to epibatidine.

DBO-83,
a 3,8-diazabicyclo[3.2.1]octane derivative, is a novel analgesic agent that
like epibatidine is a full agonist at a4b2 and ganglionic nAChRs but
unlike epibatidine DBO-83 lacks any appreciable activity at neuromuscular
junction nAChRs [30].

SIB-1508Y
is a pyridine-modified nicotine analog that is more potent and selective than
nicotine at the human a4b2 relative to the human a2b4 nAChR subtype but is significantly less potent
than nicotine at the human a7 and a3b4 subtypes.The enhanced functional subtype selectivity
of SIB-1508Y relative to nicotine is supported by neurotransmitter release
studies demonstrating differential effects of SIB-1508Y and nicotine on dopamine
and norepinephrine release.SIB-1508Y
is active in preclinical rodent and primate models of Parkinson’s disease
and is in clinical development for this indication [56].

RJR-2403
(transmetanicotine) is as potent and efficacious as nicotine in stimulating
cation efflux from rat thalamic synaptosomes (thought to reflect activation
of the a4b2 nAChR) but is 10-30-fold
less potent than nicotine in stimulating the release of dopamine from rat
striatal slices.In vivo, RJR-2403 possesses cognitive enhancement activity at least
comparable to that of nicotine but is 10-30-fold less potent in eliciting
changes in cardiovascular parameters and locomotor activity [5].

GTS-21
(4-dimethylaminocinnamylidene anabaseine; DMXB) appears to act as a potent
partial agonist at the rodent a7 subtype but is a very weak partial agonist at the rodent a4b2 subtype, blocking the effects
of ACh in a non-competitive manner [57].In contrast,
the compound appears to have very weak (12% efficacy of nicotine) and negligible
agonist activity at the human a7 and a4b2 subtypes, respectively.GTS-21 exhibits cytoprotective effects and improves cognitive performance
in preclinical models.

A-85380
is a 3-pyridyl ether that displays marked binding potency (Ki = 50 pM) and
selectivity for the a4b2 nAChR subtype relative to the a7 (Ki = 148 nM) and abdg (Ki= 314 nM) nAChR subtypes.In functional models, A-85380 is more potent
than nicotine in activating a number of rodent and human nAChR subtypes [81].Thus, A-85380
retains the high potency of epibatidine towards the a4b2 nAChR but displays a selectivity
for this subtype not observed with epibatidine.These features have made this compound a very
useful tool to probe the structure and function of this subtype.

NEURONAL
NICOTINIC RECEPTOR PHARMACOLOGY

The
multiplicity of CNS actions of (-)-nicotine in
vivo may be related to the subunit combination on the nAChR (i.e. receptor
subtype) activated, the neuronal system affected (e.g. dopaminergic vs. noradrenergic)
in a brain region mediating a specific behavior, and the intrinisic channel
properties of the subtype activated (e.g. ion selectivities & channel
conductance properties).

Functional
And Behavioral Effects Of (-)-Nicotine

Activation of nAChRs produces a variety of behavioral
and physiological effects in experimental animals, including effects on cognitive
performance, vigilance, locomotor activity, body temperature, respiration,
cardiovascular function, EEG activity, cortical blood flow, and pain perception.Many of these same actions have also been observed in humans.The diverse and often profound effects produced by compounds acting
at nAChRs are somewhat surprising given the relative scarcity of nAChRs in
the brain.Moreover, it has been difficult
to demonstrate that these CNS effects are mediated by nicotinic actions similar
to the fast excitation observed in autonomic ganglia and in striated muscle.An alternative view of central nicotinic cholinergic transmission holds
that the actions of (-)-nicotine and other nAChR activators are mediated through
modulation of other neurotransmitter systems [70].(-)-Nicotine
interacts with presynaptic nAChRs to facilitate the release of a variety of
neurotransmitters, including ACh, dopamine (DA), norepinephrine (NE), serotonin
(5-HT), g-aminobutyric acid (GABA) and glutamate [37], many of which have been implicated in mediating / modulating
a number of behaviors.

Because the addictive properties of (-)-nicotine have
been tentatively linked to interactions with the DA system (see below), the
mechanisms of nAChR-mediated dopamine release have been most extensively studied.(-)-Nicotine can induce release of DA through actions in either cell
body or terminal regions of DA neurons [37].For example,
several nAChR agonists evoke release from both striatal slices and synaptosomal
preparations in vitro[16,37,39].The observation
of release from synaptosomes suggests that at least a part of this release
is mediated by presynaptic nAChRs located directly on DA neuron terminals.The identity of the nAChRs involved is uncertain,
with data available that would support the involvement of either a4- or a3-containing receptors.The complex pharmacology of this effect has
been confirmed by the recent finding that the a3b2-selective a-conotoxin MII, blocks approximately
35-50% of the release from striatal synaptosomes but only 25% of the release
from slices [37,39].Neither the
a7 selective a-conotoxin ImI nor a-bungarotoxin affects DA-release from striatal
synaptosomes, so it is unlikely that these effects are mediated by presynaptic
a7 nAChRs on dopaminergic terminals
[39].Thus, it appears
that a3b2-containing receptors in the terminal field
play a role in DA release, along with at least one other nAChR subtype.The likelihood that an additional b2-containing nAChR is involved is suggested
by the inability of (-)-nicotine to effect measurable DA release in vivo in genetically altered mice lacking
the b2 subunit [64].The difference
between the effect of a-conotoxin MII in synaptosomes
and slices indicates that nAChRs that are not located on dopamine terminals
likely also play a role, an interpretation consistent with the observation
that the NMDA receptor antagonist kynurenic acid attentuates nAChR-mediated
DA release from striatal slices but not from striatal synaptosomes [37].

Evaluation of the mechanisms underlying nAChR-mediated
DA release reaches new levels of complexity when actions in the DA cell body
regions are considered.For example,
administration of mecamylamine directly into the VTA blocks the ability of
systemically-administered (-)-nicotine to increase DA release in the n. accumbens
[37].Conversely,
DA release in the accumbens produced by systemic (-)-nicotine can be mimicked
by administration of (-)-nicotine into the VTA [37], and DA neurons in VTA slices respond to application
of (-)-nicotine, an effect that is not observed in b2 knockout mice [64].However, it
is not likely that all of the effects of (-)-nicotine in the VTA are mediated
through direct activation of DA neurons.There is at least a component of the effect that can be blocked by
NMDA receptor antagonist administration into the VTA, suggesting that nAChR-mediated
glutamate release may be involved [76].

Examination of nAChR-mediated release of other neurotransmitters
has been less extensive, but there is pharmacological evidence that nAChR
subtypes involved in the release of NE may be distinct from those involved
in DA release [16,39].a-Conotoxin MII, for example, is much less effective
in blocking (-)-nicotine-induced NE release from hippocampal synaptosomes
than it is in blocking DA release from striatal synaptosomes [39].Thus, it appears
that NE release in the hippocampus is not modulated by presynaptic a3b2-containing nAChRs.Instead, there is evidence a3b4 receptors may be more important
for synaptosomal NE release than for DA release [16,53].Hippocampal
NE release can also be increased by direct injection of nAChR agonists into
the locus coeruleus (LC).Interestingly,
however, the NE nuclei A1 and A2, which project to the paraventricular nucleus
(PVN) of the hypothalamus, are even more sensitive to (-)-nicotine [55].NE released
in the PVN by even low doses of (-)-nicotine stimulates release of corticotropin-releasing
hormone (CRH).Thus, nAChR-mediated
NE release in the CNS has important influences on the hypothalamic-pituitary-adrenal
(HPA) axis [55].A still different
pharmacology has emerged for nAChR-mediated feedforward release of ACh that
implicates a4b2-and a7-containing nAChRs in ACh
release [37,70,82].

There is also evidence for nACh-mediated release of
the major inhibitory and excitatory neurotransmitters, GABA and glutamate.Because release of glutamate has been difficult
to detect, most of the evidence for nAChR-mediated glutamate release in the
CNS comes from analysis of electrophysiological data.Glutamate is the likely transmitter in the
connection between the medial habenula and the intrapeduncular nucleus, and
application of (-)-nicotine potentiates glutamatergic transmission at intrapeduncular
synapses with a pattern consistent with enhanced release of glutamate from
the presynaptic element [70].Electrophysiological
evidence of nAChR-mediated glutamate release has also been obtained in the
hippocampus [1,67].The pharmacology
of these effects is consistent with actions mediated through a7-containing nAChRs located on glutamatergic
terminals.Similar evidence for nAChR-mediated
GABA release has been observed, with at least a portion of this release being
mediated by a7 nAChRs [29].However, an
important role for b2-containing nAChRs (perhaps
a4b2) in mediating GABA release
has also been demonstrated [1,45].

Although the data clearly indicate that activation
of nAChRs influences the release of a multitude of neurotransmitters, it is
not entirely clear what role cholinergic regulation of transmitter release
plays in normal brain function.However,
the capacity to modulate neurotransmission and the HPA axis so broadly clearly
provides a mechanism for amplifying the impact of manipulations of the cholinergic
system.Moreover, the emerging evidence
for subtype selective regulation of neurotransmitter release suggests that
it may be possible to develop selective nAChR agents that would target specific
neurotransmitters or even specific anatomical subsystems involved in neurological
and psychiatric disease.

Therapeutic
Targets

Clinical
research with nAChR agonists has, to date, been limited primarily to the pharmacological
evaluation of (-)- nicotine; and although preclinical research continues to
focus on (-)-nicotine and other related, naturally occurring, alkaloids, a
number of novel nAChR agonists have been synthesized in the last few years
that may have therapeutic potential in a number of neurological and psychiatric
conditions.

Cognition Enhancement.Studies with both humans and experimental animals suggest that(-)-nicotine has cognition-enhancing properties, although positive
effects are not observed in all models [47,48,60].Activation
of nAChRs can enhance release of several neurotransmitters believed to be
important modulators of learning and memory, and it appears that the effects
of nAChR activators on cognitive function may be mediated through influences
on some of these neurotransmitter systems (see [10,22]).Moreover,
nAChRs may play a more direct role in information storage through modulation
of glutamatergic neurotransmission and resulting effects on synaptic plasticity
as exemplified by long term potentiation [14].It is likely
that different features of (-)-nicotine’s cognitive effects are mediated by
distinct effects on different neurotransmitter systems and that multiple nAChR
subunits are involved in the cholinergic influences on cognitive function.DHbE, n-BTX, and MLA can all disrupt
performance when injected directly into the brain (see [10,48]), potentially supporting the involvement of several
nAChR subtypes; but initial studies with b2 knockout mice did not reveal
gross memory deficits.The b2-knockout mice, however, were insensitive to
the memory-enhancing effects of (-)-nicotine [14], suggesting that the effects of (-)-nicotine on this
task require activation of b2-containing nAChRs but that
these receptors are not required for normal cognitive function.However, these mice do develop cognitive deficits
relative to wild type mice as they age, perhaps because they eventually lose
the capacity to compensate for the lack of b2-containing nAChRs [14].

The
major target disease for a cognition enhancer is Alzheimer’s disease (AD).In AD brain tissue, cortical nAChRs are markedly
reduced [77], reflecting the cholinergic deficits associated with
AD.Pilot trials using nicotine patches
have demonstrated improved attention in AD patients [48].Moreover, pharmacoepidemiological
studies have shown a reduced incidence of AD in populations of individuals
who have previously smoked [42].The potential
protective effects of (-)-nicotine in this neurodegenerative disease may be
related to neuroprotective properties observed with nicotine and other nAChR
activators in in vitro and in vivo experimental studies [25].

In
an effort to improve on the separation between the adverse and cognition-enhancing
effects of (-)-nicotine, several nAChR agonists have recently been synthesized,
some of which have entered clinical development for the possible treatment
of AD.GTS-21, ABT-418, SIB-1553A,
and RJR-2403 have shown promise in preclinical cognition models [2,5,56,58].

Attention-Deficit Disorder
(ADD).Attention-deficit disorder, with or without
hyperactivity, is a behavioral disorder characterized by distractibility and
impulsiveness.ADD is currently treated
with stimulants like amphetamine, methylphenidate, and pemoline, all of which
are thought to act via augmentation of DA neurotransmission.Given that nAChR agonists can enhance DA release
and appear to improve cognitive function, including attention, nAChR-targeted
compounds may represent a useful acute treatment for the deficits in attention
seen in ADD.Small, clinical studies
have demonstrated that (-)-nicotine patches produce significant improvements
in adults with ADD [48].It is likely,
however, that compounds more selective than (-)-nicotine and with improved
separation between efficacy and side effect liability will be required if
this approach is to be of widespread utility, particularly since the predominant
use of medication for ADD is in children.

Parkinson’s Disease.Parkinson’s disease (PD) is primarily a motor disorder, characterized
by tremors at rest, rigidity, bradykinesia, and impaired postural reflexes--effects
resulting from loss of DA cells in the substantia nigra--and is typically
treated with L-DOPA, which enhances DA transmission in the nigrostriatal pathway.The neuroprotective and DA-releasing properties of nAChR activators
suggest the possible use of this approach to treating PD.(-)-Nicotine can attenuate the loss of DA neurons in the substantia
nigra in rats with lesions of the nigrostriatal pathway, suggesting that nAChR
agonists may have the potential to protect against degeneration of this system.Acute symptom relief can be obtained with (-)-nicotine administration,
which would be consistent with the ability of this compound to increase dopamine
release [4].Promising preclinical
evidence has been obtained with SIB-1508Y, a novel nAChR agonist that increases
striatal DA release in rats more effectively than (-)-nicotine.This compound appears to have an improved preclinical safety profile
relative to (-)-nicotine and significantly potentiates the effects of L-DOPA
on motor and cognitive function in an primate MPTP model of PD [56].Moreover, since
cognitive decline is a common feature of PD, the potential cognitive-enhancing
properties of nAChR-targeted compounds may be of additional benefit [60].

Schizophrenia. Attentional deficits and
increased sensitivity to auditory stimuli in schizophrenics and their immediate
relatives may be related to a diminished gating of an auditory evoked potential
wave designated as P50 in humans and N40 in rats [46]. In normal subjects, paired presentation of auditory
stimuli results in a diminished response to the second stimulus.In schizophrenics this auditory gating is impaired.In rodents, the N40 wave originates in the hippocampal CA3
region; and auditory gating is disrupted by fimbria-fornix lesions that disrupt
hippocampal cholinergic input and by a-BgT, but not by mecamylamine.Interestingly, hippocampal tissue from schizophrenics is deficient
in a-BgT binding sites and in a7 mRNA.Among psychiatric patients, those with schizophrenia
are more likely to be smokers than those with other psychiatric diagnoses
[24].Administration
of (-)-nicotine to non-smoking relatives of schizophrenics can restore the
deficient P50 sensory gating, although this is a short - lived
effect, possibly due to nAChR desensitization.In an animal models of sensory gating deficits, (-)-nicotine and ABT-418
have short-lived effects, whereas GTS-21, a partial agonist at a7-containing nAChRs, is effective
upon repeated administration [46,79].Moreover, since
currently used antipsychotics target the DA system, it is possible that nAChR-mediated
modulation of DA neurotransmission may have therapeutic potential.The possibility that nAChR agonist effects
on cognitive function might be of benefit in schizophrenia is also worth exploring.

Tourette's Syndrome.Classical neuroleptics are used to treat Tourette's syndrome, a
condition characterized by uncontrolled spontaneous motor and verbal tics,
but are limited in their usefulness due to sedation, exacerbation of learning
difficulties, and potential tardive dyskinesia liability. (-)- Nicotine can
potentiate the behavioral effects of neuroleptics like haloperidol in a number
of preclinical models of behavior and thus may be useful in potentiating the
beneficial actions of neuroleptics while diminishing their side effect profile
[74].Pilot clinical
trials have indicated that both (-)- nicotine gum and patches can ameliorate
the symptoms of Tourette's syndrome in non-smoking adolescents who are not
satisfactorily controlled with neuroleptics [74].It is perhaps
surprising the (-)-nicotine, which releases DA, would have effects mimicking
those of DA antagonists.One possible
explanation of this apparent anomaly is that prolonged exposure to (-)-nicotine,
as would be the case with administration via a patch, might actually act by
desensitizing nAChRs.This interpretation
could account for the observation that the ameliorative effects of (-)-nicotine
patches in this condition often last long after patch removal and is supported
by the more recent finding that similar improvements can be produced by the
nAChR antagonist, mecamylamine [75].

Smoking Cessation. Tobacco smoke contains a large variety of substances; however, the addictive
nature of smoking is attributable to the actions of nicotine [80].Nicotine addiction
is a complex phenomenon involving cognition enhancement, psychological conditioning,
stress adaptation, reinforcing properties, and relief from the withdrawal
syndrome.The mesolimbic dopaminergic
system appears to play a major role in the reinforcing properties of (-)-nicotine.As is true of other addictive drugs, such as cocaine, morphine, and
amphetamine, (-)-nicotine increases glucose utilization and releases DA in
the rat nucleus accumbens [66], a region believed to be an important component of the
reward system of the brain.Moreover,
concentrations of(-)-nicotine similar
to the plasma concentrations found in smokers increase activity in DA neurons
in VTA slices [65], whereas nicotine withdrawal in rats is accompanied
by an attenuation of brain reward mechanisms [27].DA also appears
to be involved in the reinforcing properties of (-)-nicotine in smokers, although
DA antagonists increase smoking behavior in humans but decrease self-administration
in rats, an apparent discrepancy that may be related to an attempt in humans
to overcome the effects of the DA blocker [72].

The severe health liabilities and high mortality rates
associated with tobacco usage, have resulted in major efforts to identify
therapeutic treatments, most notably that of nicotine replacement therapy.Nicotine gum and nicotine patches have been
developed as aids in smoking cessation.The
initial optimism of a “cure” for smoking via nicotine replacement therapy
in the form of gum and patches has been dampened by patient disillusionment
due to the inability of either nicotine formulation to replace the nicotine
provided in cigarettes as well as overcome the psychological cues associated
with smoking, e.g. smoke inhalation and oral and hand cues.Nonetheless, second generation nicotine replacement
therapy is focused on increasing the amount of (-)- nicotine being delivered
by gum or patch and on alternative delivery systems (e.g., nasal spray, inhalers)
that more closely resemble the kinetics of nicotine administration produced
by smoking, although preliminary data do not demonstrate a clear advantage
for these alternative delivery systems [40].

Alternative
therapies under development are the“non-nicotine”
nAChR agonists and partial agonists with reduced side effect liability, as
well as combined agonist/antagonist treatment.(-)-Lobeline, a nAChR ligand with full agonist,
partial agonist and full antagonist properties depending on the test paradigm
examined, is in Phase III clinical trials for smoking cessation [31].The use of
partial agonists in drug dependence therapy combines both substitution (agonist)
and blockade of reinforcement (antagonist) in a single molecule, a concept
that has been argued to “insulate" the addicted individual from reinforcement
while preventing withdrawal symptoms.This combined agonist/antagonist concept has been validated in a
recent randomized, double-blind, placebo-controlled trial that evaluated concurrent
orally administered mecamylamine with (-)-nicotine skin patch treatment for
smoking cessation [71].

Anxiety Disorders. (-)-Nicotine has anxiolytic
actions in man and some, but not all, preclinical models of anxiety [4].Evaluation
of the human data are difficult, however, because these studies are typically
conducted with smokers and the reported anxiolytic actions of (-)-nicotine
may be confounded by relief of withdrawal-induced anxiety.Of course, the observation that (-)-nicotine-induced
withdrawal produces what might be characterized as “rebound anxiety” may be
taken as evidence that (-)-nicotine does indeed have anxiolytic effects.One interesting explanation of the anxiolytic effects of (-)-nicotine
holds that (-)-nicotine acts by desensitizing the stress response.The acute physiological effects of (-)-nicotine resemble those produced
by stress, but these effects are altered by prolonged exposure [4].Thus, (-)-nicotine
effects on stress and anxiety may be the result of receptor desensitization,
which would be consistent with the observation that continuous infusion of
(-)-nicotine at doses that would be expected to desensitize nAChRs still has
anxiolytic-like effects in experimental animals [11].

Depression. Clinical studies have demonstrated
a positive correlation between nicotine-dependence and major depression. This
is apparently not a causal relationship but results from shared predispositions
involving genetic or environmental factors [9]. One interpretation of these data is that people with
major depression use (-)-nicotine as a form of self medication, which is consistent
with the increased likelihood of depressive episodes observed during attempts
to stop smoking.There are also pilot
clinical data suggesting that transdermal administration of (-)-nicotine produces
antidepressant effects in nonsmokers [73].Effects of
(-)-nicotine on 5-HT and NE might provide a mechanism by which antidepressant
effects could be mediated, and the proposed stress-reducing effects of (-)-nicotine
and influences on the HPA axis could also play an important role [4,55].Additional
investigation of this area is clearly needed to evaluate the potential of
nAChR-targeted compounds as antidepressants.

Analgesia. (-)-Nicotine has long been
know to have antinociceptive actions in experimental animals and in man, but
the relatively short duration and modest efficacy of this effect, coupled
with the side effect profile of (-)-nicotine have discouraged development
of this approach to analgesia.However,
the discovery that epibatidine, a potent and highly efficacious antinociceptive
agent in rodent, produces its effects through actions at nAChRs has sparked
considerable interest in the potential of ChCMs as analgesics.With little separation between antinociceptive
and toxic doses in rodent models, epibatidine lacks the selectivity that would
make it a clinical candidate [3].The potential
to develop safer antinociceptive nAChR agonists, however, is exemplified by
ABT-594.This compound displays the
broad spectrum of antinociceptive activity and the full efficacy of epibatidine
in preclinical models but with an improved safety profile [23].

Further
refinements in the ChCM approach to analgesia will require an improved understanding
of the mechanisms involved in nAChR-mediated antinociception.It is likely that activation of a variety of other neurotransmitter
systems with inhibitory influences on pain signaling plays an important role
in the antinociceptive effects of nAChR agonists.Intrathecal administration ofmAChR,
5-HT, a-adrenergic, but not opioid, antagonists can
attenuate the antinociceptive effects of nAChR activation [36,69].Similarly,
lesions that deplete NE or 5HT attenuate nAChR-mediated antinociception [23,68].Given that
interference with any single one of these other neurotransmitters is insufficient
to produce complete blockade of the effect [36,69], it appears that release of several neurotransmitters
contribute in parallel rather than in series.Since intrathecal mecamylamine only modestly attenuates the antinociceptive
effects of systemic (-)-nicotine and direct injection of any of several nAChR
agonists into the brainstem can produce antinociception [23,36,69], it appears likely that activation of descending inhibition
originating in brainstem sites, such as the nucleus raphe magnus, plays an
important role in nAChR-mediated antinociception.

Relatively
little is known about the nAChR subtypes that might be involved in antinociception.The ability of DHbE to block nAChR-mediated antinociceptive effects
suggests that b2-containing (perhaps a4b2) nAChR subtypes may play
a role, whereas the lack of effect with lower doses of MLA makes it likely
that a7-containing nAChRs do not [20,38,68].Adding to the
complexity is the finding that nAChR agonists such as epibatidine can produce
behavioral signs of both irritation and antinociception when they are injected
intrathecally and that the pharmacology of these two actions are distinct
[38].This raises
the possibility that nAChR activation induces release of both nociceptive
neurotransmitters (e.g., glutamate) and antinociceptive neurotransmitters
(e.g., 5-HT and NE).Since these effects
are likely mediated by actions at different nAChR subtypes, it may be possible
to improve on the efficacy of epibatidine by developing compounds that selectively
increase the release of inhibitors of pain signaling.

Future
Aspects

The
pentameric structure of the neuronal nAChR and the considerable molecular
diversity in subunits offers the possibility of a large number of nAChR subtypes,
which, based on pharmacological precedent, may serve a variety of discrete
functions within the CNS and thus represent novel targets for therapeutic
agents. To capitalize on this opportunity, given the paucity and conflicting
nature of therapeutic data to date, will require characterization of functionally
relevant subunit combinations with respect to their localization within the
CNS and identification of selective ligands that modulate receptor function
as both direct and allosteric agonists and antagonists. An
increase in the number of pharmacophores active at nAChRs over the last few
years provides a wealth of interesting tools to complement the molecular diversity
of the receptor.This development should increase our knowledge
of the functional importance of nAChR subtype diversity.Consequently, the potential for developing
nAChR ligands for use in AD, PD, smoking cessation, anxiety, depression and
schizophrenia, as well for use as novel analgesics, appears high.Thus, nAChR pharmacology provides a challenge
and an emerging therapeutic opportunity that is comparable in many ways to
the identification and development of selective ligands with demonstrated
therapeutic utility for the ever expanding serotonin receptor superfamily.